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Abstract:

A delivery device for a active agent comprises nanoparticles based on a
biopolymer such as starch. The delivery device may also be in the form of
an aptamer-biopolymer-active agent conjugate wherein the aptamer targets
the device for the treatment of specific disorders, such as cancer. The
delivery device survives for a period of time in the body sufficient to
allow for transport and uptake of the delivery device into targeted
cells. The degree of crosslinking can provide a desired release profile
of the active agent at, near or inside the target cells. The
nanoparticles may be made by applying a high shear force in the presence
of a cross linker. The particles may be predominantly in the range of
50-150 nm and form a colloidal dispersion of crosslinked hydrogel
particles in water.

Claims:

1. A delivery system comprising, nanoparticles comprising a mass of
crosslinked biopolymers, wherein the degree of crosslinking of the
biopolymers provides a release profile of an active agent from the
nanoparticle within a predetermined range of release profiles.

2. A delivery system comprising, nanoparticles comprising a mass of
complexed biopolymers having a swell ration between about 2 and 20, and
an active agent.

3. The delivery system of claim 1 wherein the nanoparticles have number
average in a size range of 50 to 150 nm when measured by any of SEM, NTA
or DLS.

4. The delivery system of claim 1, further comprising targeting molecules
that are attached to the nanoparticles.

5. The delivery system of claim 3, wherein the targeting molecules are
selected from the group of antibodies, ligands and aptamers.

6. A delivery system of claim 3 having a ratio of glucose repeating units
to the targeting molecule, wherein the ratio is within a range between
about 100:1 to less than 1000:1.

7. The delivery system of claim 1, wherein the active agent comprises a
drug.

8. The delivery system of claim 7, wherein the drug is a chemotherapeutic
drug.

9. The delivery system of claim 8 wherein the drug is selected from the
group consisting of doxorubicin
((7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11--
trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-di-
one), cyclophosphamide
((RS)--N,N-bis(2-chloroethyl)-1,3,2-oxazaphosphinan-2-amine 2-oxide) and
carmustine (N,N'-bis(2-chloroethyl)-N-nitroso-urea).

10. A process for making a delivery system comprising: producing a
plurality of batches of crosslinked biopolymer nanoparticles, wherein
each batch has a different degree of crosslinking; loading an amount of
an active agent into the crosslinked biopolymer nanoparticles of the
plurality of batches; defining a release profile, wherein about half of
the amount is released from the biopolymer nanoparticles in a first time
period; and selecting the batch that matches the defined release profile.

11. A delivery system comprising, particles comprising a mass of
biopolymers, the biopolymers comprising glucose repeating units, and
targeting molecules, wherein a ratio of the glucose repeating units to
the attached targeting molecules is within a range between about 100:1 to
less than 1000:1 or between about 100:1 to 750:1.

12. The process of claim 10, further comprising modifying the crosslinked
biopolymer nanoparticles of the selected batch with a modifying agent so
the crosslinked biopolymer nanoparticles have a negative zeta potential.

13. The process of claim 12, wherein the modifying agent is a water
soluble oxidation catalyst.

14. The process of claim 12, wherein the modifying agent is an
immobilized oxidation catalyst.

15. The process of claim 12, wherein the modifying agent is
2,2,6,6-tetramethylpiperidin-1-oxyl radicals.

16. The process of claim 10, including the step of attaching a targeting
molecule to the crosslinked biopolymer nanoparticles in the selected
batch.

17. The process of claim 16, wherein the targeting molecule is selected
from an antibody, a ligand and an aptamer.

18. The process of claim 16, wherein the targeting molecule is an aptamer
that is adapted to target cells requiring treatment by the active agent.

19. The process of claim 10, wherein the amount of active agent is
greater than an effective amount to kill a cancer cell.

20. The process of claim 19, wherein the amount of active agent is at
least twice as great as an effective amount to kill a cancer cell.

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. (canceled)

29. (canceled)

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

Description:

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This patent claims the benefit of International application number
PCT/US2011/063102 filed on Dec. 2, 2011; U.S. patent application Ser. No.
13/310,287 filed on Dec. 2, 2011; US provisional patent application Nos.
61/653,636 filed on May 31, 2012 and 61/656,313 filed on Jun. 6, 2012
which are incorporated by reference.

FIELD

[0002] This specification relates to a delivery device for drugs or other
agents, to methods of making and using the delivery device, and to the
treatment of cancer.

BACKGROUND

[0003] The following discussion is not an admission that anything
described below is common general knowledge.

[0004] U.S. Pat. No. 6,340,527 to Van Soest et al. describes
microparticles having a particle size of 50 nm to 1 mm consisting of a
chemically crosslinked starch shell containing an active ingredient. The
particles are obtained by first preparing an oil in water emulsion of the
active ingredient in a hydrophobic phase and starch, or a dispersion of a
solid active ingredient and starch in water. The active ingredient may be
a medicament which is released in the digestive tract when the starch
degrades.

[0005] US Patent Application Publication US 2008/0241257 to Popescu et al.
describes a nanoparticle of a biodegradable polymer containing a
hydrophilic cationic drug such as streptomycin. The biodegradable polymer
may be chitosan. A pharmaceutical preparation containing the
nanoparticles is administered to a patient orally and the nanoparticles
release the drug in vivo. The drug can be complexed with a naturally
occurring polymer, such as dextran sulfate. The drug, optionally
complexed, is mixed with the biodegradable polymer followed by an
inorganic polyanion to form the nanoparticle. In one example, the
nanoparticles were about 560 nm in average size, had a zeta potential of
about +54 mV and were used to treat tuberculosis in mice.

[0006] U.S. Pat. No. 7,550,441 to Farokhzad et al. describes a conjugate
that includes a nucleic acid ligand bound to a controlled release polymer
system contained within a pharmaceutical compound. Some examples of the
polymer system are based on poly(lactic) acid (PLA) and have mean
particle sizes ranging from 137 to 2805 nm. The ligands have an affinity
for a target and are prepared through the Systemic Evolution of Ligands
by Exponential Enrichment (SELEX) process.

[0007] US Patent Publication 2009/0312402 to Contag et al. describes
nanoparticles with encapsulated nucleic acid. The polymer may be PLA, PLG
or PLGA and PEG. The particles may have ligands or antibodies attached to
them for targeting the nanoparticles to a site of interest. The
nanoparticles may have a polymer coating to provide controlled release.
The particles are in the size range of about 50 nm to about 500 nm, with
most of them in the sub-200 nm range.

[0008] US Patent Publication 2011/0244048 to Amiji et al. describes a
method of making a nanoparticle comprising combining an aqueous solution
of a solubilized therapeutic agent with a water-soluble polymer
comprising polyethylene glycol (PEG) and a fatty acid. These components
self assemble into a nanoparticle. Various dextran based particles have
means sizes ranging from 14 nm to 430 nm. The therapeutic agent may be
doxorubicin.

[0009] U.S. Pat. No. 8,048,453 to Sung et al. describes nanoparticles of
chitosan, poly-glutamic acid, and an active agent. The particles have a
mean particle size between about 50 nm and 400 nm. The active agent may
be insulin for the treatment of diabetes or an active for treating
Alzheimer's disease. The nanoparticles may be freeze-dried and loaded
into a capsule for oral administration.

INTRODUCTION TO THE INVENTION

[0010] The following introduction is intended to introduce the reader to
the invention and the detailed description to follow and not to limit or
define the claims.

[0011] This specification describes a nanoparticle based delivery device.
The device may be used for the treatment of various indications or for
other purposes. However, this specification will primarily describe the
use of the device to deliver chemotherapeutic drugs, for example, for the
treatment of cancer.

[0012] The delivery device described in this specification includes a
nanoparticle that is made predominantly from a biopolymer, for example a
starch comprising amylose, amylopectin or both. The biopolymer may have
its crystal structure broken, for example by shear forces and intensive
mixing in the presence of a hydroxilic solvent, or by other methods.
After the crystal structures have been broken, a crosslinking agent is
added to stabilize the resulting biopolymer nanoparticles. The resulting
nanoparticles comprise, for example, crosslinked high molecular weight
starch polymer that can be handled as dry agglomerated particles. The dry
particles can be dispersed in an aqueous medium to produce a stable latex
dispersion of crosslinked hydrogel nanoparticles.

[0013] The inventors believe that these crosslinked biopolymer
nanoparticles have attributes that make them useful as a drug delivery
device. In an aqueous medium, the crosslinked biopolymer nanoparticles
form a stable dispersion of swollen crosslinked biopolymer hydro-colloid
particles. The crosslinked biopolymer nanoparticles swell by taking water
into the core of the particle. This mechanism may be used to load an
active agent, into the core of the crosslinked biopolymer nanoparticles.
Loading of the active agent into the core of the crosslinked biopolymer
nanoparticles also allows unloading, or release, of the drug, for example
at, near or within target cells. In one example, the active agent is a
chemotherapy drug that is released at, near or within cancer cells.
Optionally, the crosslinked biopolymer nanoparticles that are loaded with
the drug can be administered as a liquid suspension or dried to produce a
powder.

[0014] One useful attribute of the crosslinked biopolymer nanoparticles is
that they can be broken down by chemical and enzymatic elements, but they
may persist in the body long enough to provide a sustained release of the
active agent. While native starch particles would typically survive for
less than 30 minutes in the body, starch-based crosslinked biopolymer
nanoparticles have a considerably longer half-life. In a related
attribute, the nanoparticles may provide two mechanisms for releasing a
loaded active agent. According to a first mechanism, the active agent is
released from a generally intact crosslinked biopolymer nanoparticle.
According to a second mechanism, the crosslinked biopolymer nanoparticle
can degrade and release more of the drug. This second mechanism provides
a sustained release of the drug, which is useful for active agents that
require several hours or more of residence time for an optimal effect.

[0015] Another attribute of the crosslinked biopolymer nanoparticles is
that the biopolymers are compatible with the body and ultimately can be
reabsorbed. The biopolymers and their metabolites are nontoxic. In
contrast, some synthetic polymers can cause side effects when used as a
drug delivery device. For example, polyanhydride copolymers used for drug
delivery have been associated with tissue inflammation and an enhanced
rate of infections. These side effects may be due to the synthetic
copolymers degrading via hydrolysis and yielding acidic functionalities.
Starch, however, is ordinarily a food source and can be taken up by the
body and degraded essentially without complications.

[0016] Another useful attribute of the crosslinked biopolymer
nanoparticles is their size, and the narrow particle size distribution
range within a given sample. In particular, the crosslinked biopolymer
nanoparticles are predominantly in the range of 50-150 nm. Particles
outside of this size range may be removed from the body through passive
processes, such as through capillary wall passage, or more active
processes, such as by the reticuloendothelial system (RES).

[0017] Yet another useful attribute of the nanoparticles is that the
biopolymers may be functionalized. For example, amylose and amylopectin
molecules may be oxidized and provided with carboxyl functionalities. In
this example, the functionalized, crosslinked biopolymer nanoparticles
have a more negative zeta potential which aids in the loading of some
active agents. Optionally, the functionalizing reactions may allow
attaching of targeting molecules, such as antibodies or ligands, to the
crosslinked biopolymer nanoparticles. For example, the targeting molecule
may be an aptamer that attaches, for example via a carbodiimide linkage,
directly to the surface of a crosslinked biopolymer nanoparticle. The
selection of specific aptamers, for example nucleotide or peptide
aptamers, may direct, or facilitate, interactions between the crosslinked
biopolymer nanoparticles and target cells. Other forms of
functionalization may influence the release profile of the active agent.

[0018] The inventors have further observed that the degree of crosslinking
of the starch nanoparticle influences the release profile of the active
agent from a crosslinked biopolymer nanoparticles drug delivery system.
In one example, a drug delivery system comprises biopolymer nanoparticles
that are crosslinked. The crosslinked biopolymer nanoparticles may be
functionalized to facilitate loading of an active agent and to attach a
targeting molecule, such as an aptamer. The conjugated crosslinked
biopolymer nanoparticles are loaded with an active agent. In this
example, the size of the crosslinked starch nanoparticle may provide a
longer systemic viability. The longer systemic viability may increase the
likelihood of an interaction between the targeting molecule and a target
cell. Upon a successful interaction, the drug delivery system may cross
the phospholipid bilayer and enter the target cell, through
receptor-mediated transport or otherwise. The degree of crosslinking of
the biopolymer nanoparticles may provide a desired release profile of the
loaded drug into the target cell. Optionally, the amount of attached
targeting molecule may be varied to increase or decrease the rate of
target cell uptake of the drug delivery system. Further optionally, the
crosslinked biopolymer nanoparticle may be used without a targeting
molecule.

[0019] The inventors have further observed that the amount of targeting
molecule that is attached to the crosslinked biopolymer nanoparticle
influences the uptake by the target cell.

[0020] The drug delivery system provides the ability to specifically
tailor the targeting molecule to a specific target cell, for example a
specific type of cancer cell. The rate of uptake of the drug delivery
system can also be tailored based upon the amount of the targeting
molecule that is attached. The drug delivery system provides the ability
to tailor the active agent that is delivered directly into the target
cell, for example an anti-cancer, chemotherapy drug. The drug delivery
system further provides the ability to tailor the release profile of a
tailored drug to optimize the effect of the active agent, for example, by
varying the degree of crosslinking to prolong or shorten the release
profile.

[0021] An example drug delivery device may have: 1) a nanoparticle
comprising crosslinked biocompatible or resorbable polymers, the polymers
modified after the particle was formed by chemical or enzymatic
modification, 2) an encapsulated therapeutically active agent within the
colloidal hydrogel, and, optionally, 3) an aptamer attached to the
crosslinked polymers. The nanoparticles may be colloidal hydrogel starch
particles.

[0022] A medicament described in this specification comprises a plurality
of crosslinked nanoparticles, the nanoparticles are made up mostly of
high molecular weight starch with an active agent conjugated to at least
some of the nanoparticles. Optionally, the nanoparticles may include a
targeting molecule. The medicament may be useful in the treatment of
cancer. A method of making a medicament comprises the steps of forming a
plurality of high molecular weight, starch-based nanoparticles, wherein
the nanoparticles having a size predominantly in the range of 50 to 150
nm and are crosslinked to a degree selected to provide a desired active
agent release profile and loading an active agent within the
nanoparticles. Optionally, the nanoparticles can be functionalized to
increase loading of the active agent or attach a targeting molecule or
both.

[0023] A compound described in this specification comprises a high
molecular weight starch based nanoparticle core having a size in the
range of 50 to 150 nm, a drug and, optionally, an aptamer targeting
molecule. The compound may be used for the treatment of cancer.

[0030] FIG. 6 is a schematic representation of minor swelling in an SB
latex particle and significant swelling of the crosslinked biopolymer
nanoparticles of FIG. 2 in an aqueous dispersion, illustrating the
hydrocolloid structure of the starch based nanoparticles.

[0031] FIG. 7 is a schematic model of an example crosslinked biopolymer
nanoparticle.

[0032] FIG. 8 is a chart showing the fluorescence spectrum of free
doxorubicin and doxorubicin entrapped in the example crosslinked
biopolymer nanoparticles of FIG. 2B.

[0033] FIG. 9 is a chart showing a release profile of doxorubicin from the
example crosslinked starch nanoparticles of FIG. 2B.

[0034] FIG. 10 is a chart showing the fluorescence spectrum of Calcein and
a release profile of Calcein from the crosslinked biopolymer
nanoparticles of FIG. 2B.

[0035] FIG. 11 is a schematic model of a crosslinked biopolymer
nanoparticle of FIG. 2 conjugated with a drug and an aptamer.

[0036] FIG. 12 is a chart showing the release profile of doxorubicin from
biopolymer nanoparticles with different degrees of crosslinking.

[0037] FIG. 13 is a chart that shows the uptake of crosslinked biopolymer
nanoparticles with varying levels of attached aptamer, where the captions
mean as follows: "Free aptamer" is the fluorescence result for cellular
binding or uptake of the unconjugated AS1411 aptamer; "100:1", "500:1",
"1000:1" and "5000:1" is the fluorescence result for cellular binding or
uptake of crosslinked bioconjugates formulated with relative ratios of
100, 500, 1000, and 5000 parts of glucose repeating units in the
biopolymer to one part AS1411 aptamer; and "Control 500:1" is the
fluorescence result for cellular binding or uptake of a bioconjugate
formulated with 500 parts of glucose repeating units in the biopolymer to
one part of a control aptamer sequence which is untargeted to cancer
cells.

[0038] FIGS. 14A and 14B are charts that show the viability of cells
treated with crosslinked biopolymer nanoparticles with and without a
loaded active agent.

DETAILED DESCRIPTION

Target Particle Size

[0039] Referring to FIG. 1, particle size plays a role in determining the
fate of a drug or a drug delivery mechanism after administration. Without
intending to be bound by any particular theory of operation, particles
having a size in the range of about 50 to 150 nm may enjoy longer
systemic circulation as a result of being within this size range,
independent of other properties of the particle such as surface density
or hydrophilicity which may also affect uptake by the reticuloendothelial
system (RES).

[0040] The term biopolymer nanoparticle will be used in this specification
to refer to a form of a biopolymer in which the native structure of the
biopolymer source material has been substantially removed but multiple
molecules of the bio-polymer are complexed to form discrete particles,
for example by way of cross-links between molecules within the particles.
Crosslinked biopolymer nanoparticles 10 can be made by various processes.

[0041] The presence of biopolymer nanoparticles can be determined by
observation under a scanning electron microscope (SEM); detecting
particle sizes larger than individual molecules by DLS or NTA
measurements; or, observing a maximum swelling value (alternatively
called a volume factor or swell ratio) in a very dilute dispersion of the
biopolymer nanoparticles that is less than the swell ratio of the native
or dissolved form of the biopolymer. Regarding the last technique, the
swell ratio of native starch granules is about 32 and the swell ratio of
cooked (dissolved) starch is about 44. In comparison, the swell ratio of
starch nanoparticles may be between about 2 and 20 with lower swell
ratios corresponding to more tightly cross-linked particles. A method of
determining swell ratio is described in the examples section herein and
in International Application No. PCT/CA2012/050375 which is incorporated
herein by this reference to it. Biopolymer nanoparticles useful as a drug
delivery device may have a swell ration between about 2 and 20, between
about 6 and 18 or between about 6 and 16.

[0042] Waxy corn starch is a preferred bio-based material due to its
resistance to retrograding after it has been processed relative to other
starches. Waxy corn starch also produces nanoparticles with less
cross-linker or without added cross-linker.

[0043] In one example, the biopolymer nanoparticles 10 are made according
to a process described in U.S. Pat. No. 6,677,386 (which corresponds to
International Publication WO 00/69916), which is incorporated herein by
reference. In this example process, a biopolymer feed stock, such as
starch comprising amylose or amylopectin or both, is combined with a
plasticizer. This combination is mixed under high shear forces,
preferably in a twin screw fully intermeshing co-rotating extruder, to
plasticize the biopolymer and create a thermoplastic melt phase in which
the crystalline structure of the biopolymer is removed. A crosslinking
agent is then added, while mixing continues, to form the crosslinked
biopolymer nanoparticles 10. The crosslinked biopolymer nanoparticles 10
exit the extruder as a strand of extrudate, which is ground to a fine dry
powder. The crosslinked biopolymer nanoparticles 10 are present in the
powder in an agglomerated form, and can be dispersed in an aqueous
medium. One example of crosslinked biopolymer nanoparticles 10 made by
this process is the commercially available EcoSphere® 2202 from
EcoSynthetix Inc. of Burlington, Ontario, Canada.

[0044] The biopolymer feed stock may be starch or other polysaccharides
such as cellulose and gums, as well as proteins (e.g. gelatin, whey
protein). The biopolymers may also be previously modified, e.g. with
cationic groups, carboxy-methyl groups, by acylation, phosphorylation,
hydroxyalkylation, oxidation and the like. Starch and mixtures of at
least 50% starch with other polymers are preferred. The starch, whether
used alone or in a mixture, is preferably a high molecular weight starch,
for example a molecular weight of at least 10,000, and not dextran or
dextrin. For example, the starch can contain amylose, amylopectin, or
both. Waxy starches, such as waxy cornstarch, are particularly preferred.

[0045] The following paragraphs are repeated or summarized from U.S. Pat.
No. 6,677,386 to further describe a process of making the nanoparticles.

[0046] The biopolymer preferably has a dry substance content of at least
50% by weight at the time when processing starts. Processing is
preferably done at a temperature of at least 40 degrees C., but below the
degradation temperature of the polymer, for example 200 degrees C. The
shear can be affected by applying at least 100 J of specific mechanical
energy (SME) per g of biopolymer. Depending on the processing apparatus
used the minimum energy may be higher; also when non-pregelatinised
material is used, the minimum SME may be higher, e.g. at least 250 J/g,
especially at least 500 J/g.

[0047] The plasticiser may water or a polyol (ethyleneglycol,
propyleneglycol, polyglycols, glycerol, sugar alcohols, urea, citric acid
esters, etc.). The total amount of plasticisers (i.e. water and others
such as glycerol) is preferably between 15 and 50%. A lubricant, such as
lecithin, other phospholipids or monoglycerides, may also be present,
e.g. at a level of 0.5-2.5% by weight. An acid, preferably a solid or
semi-solid organic acid, such as maleic acid, citric acid, oxalic,
lactic, gluconic acid, or a carbohydrate-degrading enzyme, such as
amylase, may be present at a level of 0.01-5% by weight of biopolymer.
The acid or enzyme assists in slight depolymerisation, which is assumed
to be advantageous in the process of producing nanoparticles.

[0048] The crosslinking is preferably at least in part reversible, i.e.
the crosslinks are partly or wholly cleaved during the mechanical
treatment step. Examples of reversible crosslinkers are a) dialdehydes
and polyaldehydes, which form more stable full acetals and reversibly
form hemiacetals, and b) anhydrides and mixed anhydrides, which form
ester linkages (e.g. succinic and acetic anhydride) and the like.
Suitable dialdehydes and polyaldehydes are glutaraldehyde, glyoxal,
periodate-oxidised carbohydrates, and the like.

[0049] Such crosslinkers may be used alone or as a mixture of reversible
crosslinkers, or as a mixture of reversible and non-reversible
crosslinkers. Thus, conventional crosslinkers such as epichlorohydrin and
other epoxides, triphosphates, divinyl sulphone, can be used as
non-reversible crosslinkers for polysaccharide biopolymers, while
dialdehydes, thiol reagents and the like may be used for proteinaceous
biopolymers. The crosslinking reaction may be acid- or base-catalyzed.
The level of crosslinking agent can conveniently be between 0.1 and 10
weight % with respect to the biopolymer. The crosslinking agent may be
present at the start of the mechanical treatment, but in case of a
non-pre-gelatinised biopolymer such as a starch with native starch
granules, it is preferred that the crosslinking agent is added later on,
i.e. during the mechanical treatment.

[0050] The mechanically treated, crosslinked biopolymer is then formed
into a latex by dispersion in a suitable medium, usually water and/or
another hydroxylic solvent such as an alcohol), to a concentration of
between 4 and 50 weight % especially between 10 and 40 wt. %. Prior to
the dispersion a cryogenic grinding step may be performed, but stirring
with mild heating may work equally well. This treatment results in a gel
which either spontaneously or after induction by water adsorption, is
broken into a latex. This viscosity behavior can be utilised for
applications of the particles, such as improved mixing, etc. If desired,
the dispersed biopolymer may be further crosslinked, using the same or
other crosslinking agents as describe above. The extrudate is
characterised by swelling in an aqueous solvent, e.g. water or a mixture
of at least 50% water with a water-miscible solvent such as an alcohol,
and by exhibiting a viscosity drop afterwards to produce a dispersion of
nanoparticles.

[0051] International Patent Application Publication No. WO 2008/022127 A2
and its equivalent US Patent Application Publication Number 2011/0042841
A1 describe a process for producing biopolymer nanoparticles in large
quantities. US Patent Application Publication Number 2010/0143738 A1
describes a process for producing biopolymer nanoparticles conjugative
with additives during the extrusion process. US Patent Application
Publication Numbers 2010/0143738 A1 describes a process for producing
biopolymer nanoparticles conjugated with additives during the extrusion
process. These publications are incorporated herein by reference.

[0052] The production of biopolymer nanoparticles similarly formed by
reactive extrusion and comprising starch essentially without crystalline
structures is described in Starch nanoparticle formation via reactive
extrusion and related mechanism study, Delong Song et al., Carbohydrate
Polymers 85 (2011) 208-214. The contents of this publication are
incorporated herein by reference. This publication is incorporated herein
by reference. Using various materials and reaction conditions,
dispersions having particles with number average particle sizes up to
about 2000 nm were produced. Various other methods of making biopolymer
nanoparticles are also summarized in this paper.

[0053] Another method reported to produce biopolymer nanoparticles by
reactive extrusion process from waxy corn starch is described in
International Publication Number WO 2011/071742 A2, Process for Preparing
Stable Starch Dispersions, by Welsch et al., published on Jun. 16, 2011.
This publication is incorporated herein by reference. This process
comprises introducing a feed starch and an hydroxylc liquid to an
extruder. Shear forces are applied in the extruder to the starch and the
liquid in the substantial absence of a crosslinker under conditions
sufficient to prepare a stable dispersion of starch particles in the
hydroxylic liquid.

[0054] Another method reported to produce biopolymer nanoparticles is
described in International Publication Number WO 2011/155979 A2, Process
for Preparing Stable Dispersions of Starch Particles, by Welsch et al.,
published on Dec. 15, 2011. The contents of this publication are
incorporated herein by reference. In this process, a feed starch and an
aqueous liquid are introduced into a rotor stator mixer. The feed starch
and aqueous liquid are maintained in the rotor stator mixer at a
temperature ranging from a gelation temperature to less than a
solubilization temperature. The feed starch is sheared into starch
particles with the rotor stator mixer to form the dispersion of starch
particles in the aqueous liquid.

[0055] Another method of producing a starch nanoparticle is described in
U.S. Pat. No. 6,755,915 to Van Soest et al. (Jun. 29, 2004) which teaches
a method of preparing starch particles with a size range of 50 nanometers
to 100 microns. The disclosure of this patent document is incorporated
herein by reference. The method includes the steps of: dispersing starch
in a first water phase; dispersing a second hydrophobic phase in the
first phase to form an oil-in-water emulsion; inverting the oil-in-water
emulsion to a water-in-oil emulsion; crosslinking the starch in the first
phase; and separating the formed starch particles. The phase inversion
can occur by including a surfactant that stabilizes a water-in-oil
emulsion or the surfactant can be temperature sensitive and increasing
the reaction temperature. The inversion can also occur by the addition of
further hydrophobic liquids or various suitable salts. In this process
the starch molecules can remain partially granular during both the
crosslinking reaction and complete gelatinisation of the granular starch
can be effected before, during or after the phase inversion.
Gelatinization occurs by increased temperature, salts or combinations
thereof.

[0056] Another method reported to produce biopolymer nanoparticles is
described in WO 2010/084088 to Santander Ortegea et al. (international
publication Jul. 29, 2010). The contents of this publication are
incorporated herein by reference. The method includes the steps of
preparing starch derivatives by a first disintegration step, with solvent
and increased temperatures, followed by common substitution methods, such
as esterification, etherification. The starch derivatives are added to an
organic solvent and an oil/water emulsion is prepared with a high shear
mixer. Sonication may be used to improve the oil droplet distribution.
The organic phase is then removed through a membrane, which results in an
aqueous dispersion of starch-based nanoparticles.

[0057] Another method of making biopolymer nanoparticles is described in
WO 2010/065750 to Bloembergen et al. which teaches that Brabender static
high shear mixers and Sigma Blade mixers may be used in place of an
extruder to produce nanoparticles by way of shearing starch granules in
the presence of a crosslinker. The contents of this publication are
incorporated herein by reference.

[0058] Alternatively, fragmented particles may be used. British patent GB
1420392, for example, describes a method of producing fragmented starch
particles that are partially cross-linked and partially crystalline or
soluble that may be used as an alternative to nanoparticles.
Nanoparticles are preferred, however, since they are likely to be less
prone to retrogradation.

[0059] The process can be operated to produce particles that have a number
average particle size in the range of 50 to 150 nm and which, considering
a distribution of their particle sizes, are also predominantly in the
range of 50 to 150 nm in size. Such particles include, for example,
EcoSphere® 2202 particles commercially available from Ecosynthetix
Inc. of Burlington, Ontario, Canada and EcoSynthetix Ltd. of Lansing,
Mich., USA. These products are made primarily from starch including
amylose and amylopectin. The product is normally sold to replace
petroleum based latex binders in industrial applications, such as coated
paper and paperboard. The product is provided in the form of a dry powder
of agglomerated nanoparticles with a volume mean diameter of about 300
microns. When mixed in water and stirred, the agglomerates break apart
and form a stable dispersion of the nanoparticles.

[0060] Comparing FIG. 2A to FIG. 2B, the EcoSphere® 2202, as an
example of crosslinked biopolymer nanoparticles 10, are about 100 to 300
times smaller than native starch granules 20. Whereas a starch granule 20
may be 15 microns in size, the nanoparticles 10 are clearly well under
200 nm in size. Accordingly, the effective surface area of the
nanoparticles 10 is much greater, for example 200 m2/g or more.

[0061] FIGS. 3 and 4 illustrate particle size measurements of an aqueous
dispersion of the EcoSphere® 2202, as an example of crosslinked
biopolymer nanoparticles 10, by Dynamic Laser Light Scattering (DLS) and
by Nanoparticle Tracking Analysis (NTA), respectively. These two
techniques are complementary, given that the NTA technique is a direct
measurement of the diffusion coefficient for individual particles tracked
via video tracking software (and relates that to particle diameter via
the Stokes-Einstein equation), and can measure particles in the range of
50-1000 nm, while DLS can measure to smaller particle sizes below 50 nm.
Other techniques, including oscillating probe Atomic Force Microscopy
(AFM), Scanning Electron Microscopy (SEM), Environmental SEM (ESEM),
Transmission Electron Microscopy (TEM) and Scanning/Transmission Electron
Microscopy (STEM), all provided similar particle size images consistent
with the data in FIGS. 3 and 4.

[0062] Referring to FIG. 3, most of the EcoSphere® 2202 particles have
a size in the range of about 50 to 100 nm. As indicated in the NTA
measurements, most of the particles (D50) are under 120 nm in size and
there are virtually no particles larger than 400 nm. Any particles larger
than 1000 nm would be removed quickly from the body causing no harm but
wasting some of an intended dosage of the drug. Accordingly, if a sample
includes material amounts of particles over 1000 nm in size, these may be
removed by filtration, or otherwise, before an active agent 22 is loaded
into the nanoparticles.

[0063] Referring to FIG. 5, the crosslinked biopolymer nanoparticles 10
are generally smaller than particles found in synthetic latex emulsions
such as styrene-butadiene (SB) emulsions, acrylic emulsions and polyvinyl
acetate (PVAc) emulsions. The crosslinked biopolymer nanoparticles 10
have a narrow size-distribution, with a polydispersity index of about
30%, and properties characteristic of polymer colloids. Since the
crosslinked biopolymer nanoparticles 10 are predominantly in the size
range of about 50 to 150 nm (for example 50% or more of the nanoparticles
by number or mass may be in this range) the crosslinked biopolymer
nanoparticles 10 may be cleared more slowly from the systemic circulation
(liver, spleen) than is the case of larger particles. The crosslinked
biopolymer nanoparticles 10 may have hydrophilic properties, which may
further inhibit removal by the RES. The degradation products of the
starch nanoparticles (D-glucose and maltodextrans) are non-toxic. The
additional natural materials and chemicals that are used to make the
starch crosslinked biopolymer nanoparticles are also relatively
non-toxic.

[0064] The crosslinked biopolymer nanoparticles 10 are not water soluble,
but instead form a stable latex dispersion of swollen hydrogel colloidal
crosslinked particles in water.

[0065] FIGS. 6A and 6B depict the latex dispersion consisting of
water-swollen crosslinked biopolymer nanoparticles 10, which can de-swell
with increasing solids. This permits dispersions that can be made at
higher solids. In contrast, the particles in synthetic latex emulsions do
not swell nor contain a substantial portion of water inside the colloid
particles. The swelling characteristics of typical SB latex and colloids
of biopolymer nanoparticles have been compared and reported in a number
of articles (see Do lk Lee, Steven Bloembergen, and John van Leeuwen,
"Development of New Biobased Emulsion Binders", PaperCon2010, "Talent,
Technology and Transformation", Atlanta, Ga., May 2-5, 2010; and, Steven
Bloembergen, Edward VanEgdom, Robert Wildi, Ian. J. McLennan, Do lk Lee,
Charles P. Klass, and John van Leeuwen, "Biolatex Binders for Paper and
Paperboard Applications", Journal of Pulp and Paper Science, 36, No 3-4,
p. 151-161, 2011; J. Y. Shin, N. Jones, D. I. Lee, P. D. Fleming, M. K.
Joyce, R. DeJong, and S. Bloembergen, "Rheological Properties of Starch
Latex Dispersions and Starch Latex-Containing Coating Colors", TAPPI,
PaperCon 2012, "Growing the Future", New Orleans, La., Apr. 21-25, 2012).
The contents of these publications are incorporated herein by reference.

[0066] FIG. 7 illustrates a schematic model for the crosslinked biopolymer
nanoparticles 10. The crosslinked biopolymer nanoparticles 10 can be
thought of as one crosslinked macromolecular unit, with --R--
representing an intermolecular crosslink between individual biopolymers
12. Other types of crosslinked structures may exist, such as
intramolecular crosslinks.

[0067] The crosslinked biopolymer nanoparticles 10 comprise a core 14 and
a shell 16. The core 14 receives and releases water as it swells and
de-swells and the shell 16 provides a steric stabilization mechanism for
the dispersed colloid particles. Water is released, bound and adsorbed
from the core 14 through the shell 16. The structure of the crosslinked
biopolymer nanoparticles 10 is further described in Steven Bloembergen,
Ian. J. McLennan, John van Leeuwen and Do lk Lee, "Specialty Biobased
Monomers and Emulsion Polymers Derived from Starch", 2010 PTS Advanced
Coating Fundamentals Symposium, Munich, Germany, Oct. 11-13, 2010.

[0068] Aqueous dispersions of the crosslinked biopolymer nanoparticles 10
are stable for up to 12 months or longer. Because typical native starches
contain very high molecular weight amylopectin polymer (millions of
daltons) and high molecular weight amylose polymer (hundreds of thousands
of daltons), solutions up to 5 or 10% solids can have very high gel-like
viscosities. Commercial dispersions of corn starch granules typically
reach up to about 30% solids or higher, because these products have been
chemically, thermally or enzymatically treated to reduce their molecular
weight in order to attain higher solids contents. This is the typical
molecular weight/solids trade off that one faces to maintain a reasonably
low viscosity for polymer solutions. Purer dispersions with higher solids
content (up to about 40% solids), and ultra-high solids formulations (up
to 72% solids) have been developed using the crosslinked biopolymer
nanoparticles 10. This may be beneficial for drug delivery applications,
where a high solids concentration facilitates loading of greater amounts
of the active agent 22 into the crosslinked biopolymer nanoparticles 10.

[0069] The crosslinked biopolymer nanoparticles 10 may be loaded with an
active agent 22, for example a drug, or other agent, and used as a
delivery device. Loading of the active agent 22 may also be referred to
as conjugating or encapsulating.

[0070] As discussed above, the core 14 of the nanoparticles takes in water
as it swells. Similarly, small molecules, such as some drugs, or other
agents can be taken up, adsorbed, or otherwise loaded into the core of
the nanoparticles. An example presented further below will describe
loading of the drug doxorubicin in the crosslinked biopolymer
nanoparticles 10 by a phase separation method (Example 1) and by ethanol
precipitation (Example 4). By itself, doxorubicin has been linked to
acute cardiotoxicity which limits its use. In other experiments,
Carmustine and BCNU (bis(chloroethylnitrosourea)) have been also been
loaded into the crosslinked biopolymer nanoparticles 10.

[0071] It can be expected that other methods of loading the drug may also
be used, and that other drugs and agents can similarly be loaded. For
example, other active agents 22 may include cyclophosphoramide and
camptothecins that may be loaded into the crosslinked biopolymer
nanoparticle 10 and, like doxorubicin, make the crosslinked biopolymer
nanoparticles 10 useful in the treatment of cancer. The crosslinked
biopolymer nanoparticles 10 may also encapsulate non-chemoactive agents,
such as antisense oligonucleotides, peptides, and cytokines for other
therapeutic applications.

[0072] After the active agent 22 is loaded, the crosslinked biopolymer
nanoparticles 10 can be recovered by lyophilization, which results in a
powder of the nanoparticles loaded with the encapsulated active agent 22.
The powder can be mixed with water, or another hydroxylic solution, to
disperse the crosslinked biopolymer nanoparticles 10 into a stable
colloidal dispersion. The dispersion can be administered to treat a
patient in the liquid form, for example orally, by intra-venous infusion
or injection. The powder can be mixed with a pharmaceutical carrier and
made into a solid or gelled drug product, such as a tablet or capsule.
The drug product may be administered in any known manner used for
pharmaceutical products, such as orally, rectally, transdermally and the
like.

[0073] The biopolymers 12 may be modified, also referred to as
functionalized, through chemical or enzymatic modifications before,
during or after forming the crosslinked biopolymer nanoparticle 10. In
principle, any chemical or enzymatic modification known for
polysaccharides can be employed. For example, a summary of various
chemical and enzymatic oxidation processes is provided in column 1, line
66 to column 3, line 50 in R. A. Jewel et al., U.S. Pat. No. 6,379,494,
"Method of Making Carboxylated Cellulose Fibers and Products of The
Method", Apr. 30, 2002, the disclosure of which is incorporated herein by
reference. Although these methods are discussed in relation to cellulose,
many if not all are adaptable to starch polymers.

[0074] In Example 4, a biopolymer 12 of starch is functionalized after the
crosslinked biopolymer nanoparticles 10 are formed. In particular, the
biopolymers 12 were oxidized to add carboxyl functional groups. While
this is described in Example 4 as relating primarily to the attachment of
a targeting molecule 18, to be discussed further below, functionalizing
the crosslinked biopolymer nanoparticle 10 may also facilitate loading of
the active agent 22.

[0075] Functionalizing the crosslinked biopolymer nanoparticle 10 by
chemical or enzymatic methods may also attach other types of functional
groups to the biopolymers to provide binding sites for the targeting
molecule 18, the active agent 22 or both. The surface of the crosslinked
biopolymer nanoparticles 10 may also be modified, chemically or otherwise
to alter systemic clearance rates to provide a better control of the
delivered active agent 22 and a targeted delivery, if any.

[0076] For example, the oxidation resulted in a change in the zeta
potential of the crosslinked biopolymer nanoparticle 10. The zeta
potential of a non-functionalized crosslinked biopolymer nanoparticle 10
is in the range of 0 to negative 6 mV. An oxidized crosslinked biopolymer
nanoparticle 10 demonstrates a zeta potential of about negative 25 mV.
The oxidation reaction may also be controlled to provide functionalized
crosslinked biopolymer nanoparticles 10 that have an intermediate zeta
potential value, for example between about negative 6 to about negative
25 mV. Tuning the zeta potential of the crosslinked biopolymer
nanoparticles 10 may allow selective loading of the active agent 22 and,
additionally, this tuning may provide control of the release profile of
the active agent 22. Many small molecules being developed for cancer
treatment are hydrophobic and lipophilic and, hence, they are difficult
to dissolve. Functionalizing, for example by oxidative modification, or
otherwise, of the crosslinked biopolymer nanoparticle 10 may enhance the
ability of the small molecule, hydrophobic/lipophilic drugs to be loaded
onto the crosslinked biopolymer nanoparticle 10.

[0077] While the water soluble TEMPO catalyst
(2,2,6,6-tetramethylpiperidine-1-oxyl radical) used in Example 4 provided
starch functionalities throughout the crosslinked biopolymer nanoparticle
10, an immobilized TEMPO catalyst causes only biopolymers 12, or portions
of biopolymers 12, located at or near the shell 16 to be functionalized.
This approach could be used, for example, to attach the targeting
molecule 18 to the surface of the crosslinked biopolymer nanoparticle 10
with less modification of the zeta potential of the core 14. Optionally,
a soluble catalyst can be used if a greater change in zeta potential is
desired.

[0078] While any form of oxidation may be used, the TEMPO oxidation is
preferred. The TEMPO catalyst is used to specifically modify the C6
hydroxyl of the glucopyranoside position to a carboxyl functionality.
This process prevents the molecular weight reduction of the
polysaccharide polymer that is common with many other oxidative
processes.

[0079] Many functionalizing techniques are known to add aldehyde groups to
polysaccharide polymers. Without intending to exclude the possibility
that one of these functionalization techniques might be useful, they are
not currently preferred. The aldehyde groups are reactive and tend to
cause the crosslinked biopolymer nanoparticles 10 to agglomerate and
stick together. This interferes with creating a colloidal dispersion, and
so may also interfere with distribution of the crosslinked biopolymer
nanoparticles 10 in the body.

[0080] As described above, the zeta potential of unmodified crosslinked
biopolymer nanoparticles 10 is low, hence the observed colloidal
stability is attributed mainly to steric stabilization. Without being
bound by theory, the shell 16 contains short polysaccharide chains which
project into the aqueous environment. These chains may function as a
colloidal stabilizer for the crosslinked biopolymer nanoparticle 10 in
water and as a partial hydrophilic shell for bound water. This in turn
may prevent or slow the release, efflux or diffusion of hydrophobic
active agents 22 from the crosslinked biopolymer nanoparticle 10.

[0081] In some of the examples provided below, doxorubicin was used as the
active agent 22. The doxorubicin was loaded into the crosslinked
biopolymer nanoparticles 10 so that the release profile could be followed
using a fluorescence technique. This work has demonstrated a biphasic
release profile with suitable release kinetics spanning multiple hours of
sustained release of the doxorubicin. The fluorescence of the
doxorubicin-loaded crosslinked biopolymer nanoparticles 10 declines over
time but some fluorescence remains even after 12 hours. This indicates
that not all of the doxorubicin is released from an intact particle. The
remainder of the loaded doxorubicin, however, will be released in the
body as the crosslinked biopolymer nanoparticle 10 degrades, for example
due to alpha-amylase enzymes. The complete release time may be 24, 48, 72
hours or more. Other drugs or compounds that are used as the active agent
22 may demonstrate a similar sustained and biphasic release profile.

[0082] Referring to FIG. 12, the inventors have observed a relationship
between the degree of crosslinking of the biopolymer 12 and the release
profile of the active agent 22. As described in Example 5, three
different batches of crosslinked biopolymer nanoparticles 10 were
produced, with relatively low, medium and high degrees of crosslinking.
The greater the degree of crosslinking of the crosslinked biopolymer
nanoparticle 10 the slower the rate of release of the active agent 22. In
contrast, the batch of crosslinked biopolymer nanoparticles 10 that had a
relatively lower degree of crosslinking demonstrated a faster rate of
active agent 22 release.

[0083] In animal studies described in Example 3, doxorubicin loaded
crosslinked biopolymer nanoparticles were used to treat glioblastoma
multiforme, a primary brain tumor in athymic mice. These studies
demonstrated a 30% increase in survival for the mice treated with
doxorubicin-loaded crosslinked biopolymer nanoparticles 10 relative to
the appropriate controls. Without intending to limit the invention to any
particular theory, this success is attributed to one or more of several
factors including the size, the surface properties, and the sustained
release kinetics of the crosslinked biopolymer nanoparticles 10. The
encapsulated doxorubicin is believed to enter the cell via endocytosis
due to the relatively small size of the nanoparticle, while the free drug
is metabolized and excreted.

[0084] FIG. 11 is a schematic of an example bioconjugate device 30
comprising a crosslinked biopolymer nanoparticle 10, a functionalized
biopolymer 12, an optional targeting molecule 18 and a loaded active
agent 22, that is shown within the core 14. FIG. 11 is merely a schematic
and is not a representation or limitation of how the active agent 22 may
be loaded in the bioconjugate device 30.

[0085] The bioconjugate device 30 may be used for the delivery of
therapeutically effective doses of the active agent 22 to targeted cells
for the treatment of specific disorders. The bioconjugate device 30 can
be made by functionalizing crosslinked biopolymer nanoparticles 10,
loading an active agent 22 within the colloidal polymer hydrogel.
Optionally, the surface of the crosslinked biopolymer nanoparticles 10
can also be functionalized by attaching the targeting molecule 18. The
targeting molecule 18 can be any antibody, ligand, signal sequence or
molecule that attaches to a functionalized biopolymer 12, for example
within the shell 16, and that is capable of increasing the bioconjugate
device 30 interaction with specified target cells. The term interaction
refers to target cell surface receptor--targeting molecule 18 recognition
and bonding, or other indirect mechanisms, whereby the presence of the
targeting molecule 18 increases the likelihood of a bioconguate device 30
in the systemic circulation to target, interact with and ultimately
transport into a target cell. Fluorescence studies indicate that the
crosslinked biopolymer nanoparticles 10 can be taken into the cell
nucleus. Without intending to be limited by theory, the transport
mechanism is believed to be endocytosis, which may be receptor mediated,
or not. Optionally, the degree of crosslinking of the crosslinked
biopolymer nanoparticle 10 can be varied to provide a desired release
rate of the active agent 22 from the core 14.

[0086] In one example, the targeting molecule 18 is an aptamer that
typically has a size of less than about 10 nm and increases the diameter
of the bioconjugate device 30 by only about 20 nm or less. The aptamer is
capable of binding to a target molecule that is located in a specific
site which may include cancer cells. For example, AS1411 is an aptamer
that has been shown to bind to nucleolin (Soundararajan et al., "Plasma
Membrane Nucleolin Is a Receptor for the Anticancer Aptamer AS1411 in
MV4-11 Leukemia Cells", Molecular Pharmacology, Vol. 76, No. 5, 2009).
Binding to nucleolin receptors is useful in the treatment of a wide array
of cancers such as renal cell carcinoma, breast cancer, prostate cancer
and others. AS1411 may also be tagged with, for example, a Cy3
fluorescent tag for imaging purposes.

[0087] Another potentially useful targeting molecule 18 is the aptamer
sgc4. This aptamer was developed by way of the SELEX process from T-cell
leukemia cell lines and is able to recognize leukemia cells (Shannguan et
al., "Aptamers Evolved from Cultured Cancer Cells Reveal Molecular
Differences of Cancer Cells in Patient Samples", Clinical Chemistry 53,
No. 6, 2007). However, sgc4 has a short biological life if it is not
conjugated. Its sequence is described in US Patent Publication
2009/0117549. Shorter variants of the sequence may also be effective.
Sgc8c aptamers have also been reported to be useful for targeting
leukemia cells (Ozalp et al., Pharmaceuticals 2011, 4, 1137-1157)

[0088] Targeting molecules, such as aptamers, with an amine modification
on the 3' end of the DNA can be linked or attached, for example by one or
more covalent bonds, to the carboxyl groups of the functionalized
biopolymers 12. The linkage may be made, for example, using EDC
chemistry, or by another linkage between the carboxyl and the amine. An
example of such a linking using an amine modified test strand of DNA is
described in Example 4. Similarly, aptamers such as AS1411 and sgc4 can
also be provided with an amine modification and are expected to also
attach to functionalized biopolymers 12. When also loaded with an active
agent 22, such as doxorubicin, the resulting 22 bioconjugate device 30
may deliver therapeutically effective amounts of the active agent 22 to
targeted leukemia cells, or other cancer cells.

[0089] By using an immobilized TEMPO as the catalyst to oxidize the
biopolymer 12 forms carboxyl groups in the shell 14. These carboxyl
groups may be activated by NHS and EDC to attach an amine-modified
targeting molecule 18, such as an aptamer, to the surface of the polymer
colloid, thereby forming a covalent linkage. The number of functional
groups on the surface of the nanoparticle may determine the aptamer
surface density and, ultimately, the rate of target cell uptake.

[0090] TEMPO reacts with the hydroxyl groups on the starch polymers in an
aqueous medium to create the desired carboxyl groups (--COOH) by the
process known as TEMPO-mediated carboxylation. NaBr is used to stabilize
this reaction. Hypochlorite (NaClO) initiates the reaction by keeping the
pH at 10.2-10.5. Then HCl can be used to lower the pH and reprotonate the
carboxyl groups. 1-Ethyl-3-(3-dimethylaminopropyl) carbodimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are chemicals which
can act as coupling agents to form carboxyl-amino covalent linkages,
which link the functionalized biopolymers 12 to the 3'-amine-modified
ssDNA aptamer. In this manner, a bioconguate device 30 can be loaded with
an active agent 22 and provided with a targeting molecule 18 to increase
the interaction of the bioconjugate device 30 with target cells.

[0091] Optionally, other molecules can be attached to the crosslinked
biopolymer nanoparticle 10, through functional groups or other
modifications. For example, PEG or other passivating polymer molecules
can be attached to improve the half-life and possibly the bioavailability
of the crosslinked biopolymer nanoparticles 10 and the bioconjugate
devices 30 based thereupon.

[0092] The following examples serve to illustrate one or more parts of one
or more inventions and are not intended to limit any claim. Reference is
made in the examples to EcoSphere® 2202 from EcoSynthetix Inc. as a
non-limiting example of a crosslinked biopolymer nanoparticles 10.
However, other nanoparticles with similar properties of drug loading,
functionalization, target molecule attachment and variable degrees of
crosslinking are also contemplated.

Example 1

Incorporation of Fluorescent Agents into Starch Based Nanoparticles

[0093] Incorporation of two compounds, in particular the fluorescent model
compound Calcein and the fluorescent anticancer agent doxorubicin (IUPAC
Name: (7S,9S)-7-[(2R,4S,5S,6S)-4-ami
no-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)--
4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione; commercial products
include Adriamycin® and Doxil®), into crosslinked biopolymer
nanoparticles 10 (EcoSphere® 2202 from EcoSynthetix Inc.) was
accomplished by a phase separation technique. This technique involves the
formation of a water-in-oil emulsion. In a 250 mL round bottom flask, the
starch based nanoparticles were dispersed at <5% solids (w/w) in water
under mechanical agitation at a pH of about 10 using dilute caustic. The
resultant dispersion was titrated to a pH of 7 using dilute hydrochloric
acid. The substance to be incorporated in the crosslinked biopolymer
nanoparticle colloid matrix (calcein or doxorubicin) was dissolved in the
dispersion containing the biopolymer nanoparticles. The amount of
encapsulated active agent 22 prepared ranged from 0.04%-0.4% (w/w). The
flask was placed inside an insulated container and secured properly. The
solution was then stirred for several minutes. Hexane was added drop wise
under continuous agitation until an emulsion was formed. The emulsion was
immediately frozen using liquid nitrogen. The flask was connected to a
vacuum system and lyophilization was carried out at -85° C. After
24 hours, when the vacuum gauge indicated no further vapor removal, the
dried sample was removed from the vacuum system and stored at -10°
C.

Example 2

Drug Release Studies

[0094] The use of fluorescent dyes as spectral probes to investigate
inclusion complexation is known (see Saenger, W. Angew. Chem. 1980, 92,
343-61 and Wenz, G. Angew. Chem. 1994, 106, 851-70). This approach was
adopted in studying the efficacy of starch based nanoparticles (in this
example we used EcoSphere® 2202 from EcoSynthetix Inc.) to
encapsulate selected drugs and the ability of this material to release
the drug over time. Fluorescent compounds such as calcein and doxorubicin
are very sensitive to environmental changes. The fluorescent signal of
the molecules was enhanced when it was incorporated into the matrix of
starch based nanoparticles. As shown in FIG. 8, the signal intensity of
free doxorubicin is much lower than that of the encapsulated doxorubicin.
In addition, a significant hypochromic shift (change of spectral band
position in the emission spectrum of a molecule to a shorter wavelength)
is observed when doxorubicin is encapsulated. FIG. 9A shows a series of
fluorescence spectra of doxorubicin obtained as a function of time. It
can be seen that there is a significant decrease in signal intensity with
time, indicating sustained release of the active agent 22. In addition,
there was a relatively small bathochromic shift (change of spectral band
position in the emission spectrum of a molecule to a shorter wavelength)
observed. Without intending to be limited to any theory of operation, it
appears that the reduced shift indicates a biphasic release mechanism
given that not all of the active agent 22 was released over the course of
the 12 hour experiment. FIG. 10A shows a series of fluorescence spectra
of calcein obtained as a function of time. It can be seen that there is a
decrease in signal intensity with time, indicating sustained release of
the active agent 22.

[0095] The data shown in FIGS. 9A and 10A illustrate that enhancement in
signal intensity for calcein and doxorubicin due to inclusion
complexation with the starch based nanoparticles can be used to monitor
the release of the active agent. FIGS. 9B and 10B are plots of signal
intensity as a function of time at the maximum signal intensity of the
fluorescence emission spectra for doxorubicin and calcein, respectively.
These data show that the concentration of fluorophore molecules inside
the supramolecular cavity is changing with time. The release of the
molecules appears to be proportional to the concentration gradient of the
active agent. The sustained release of active agent from the biopolymer
nanoparticles extended to more than 10 hours. The results demonstrate
that the biopolymer nanoparticles provide a stable matrix for the steady
release of active agent over an extended time period. The release
mechanism appears to be predominantly diffusion controlled.

Example 3

In Vivo Studies of Human Xenographs Implanted in Athymic Mice

[0096] In order to demonstrate the efficacy of the crosslinked biopolymer
nanoparticles 10 as a drug delivery device, they were loaded with the
anticancer drug doxorubicin as described in Example 1. The doxorubicin
loaded in crosslinked biopolymer nanoparticles 10 was administered to
athymic mice which had a human xenograph of a primary brain tumor (D 245
glioblastoma multiform) previously grown at a subcutaneous site. Athymic
mice were chosen for these studies because normal mice are capable of
immunologically rejecting implanted foreign xenographs, specifically
human tumors. The animals (both control and treated) were monitored for
tumor regression and survival. The results of the study are presented in
Table 1.

[0097] The procedure consisted of inoculation of the tumor xenograph into
a subcutaneous site in athymic mice. The subcutaneous tumors were grown
to approximately 200 cubic millimeters in size (6-8 mm in diameter).
Subsequently, either the free drug or the drug loaded nanoparticles were
injected at the tumor site or i.p. (intra peritoneal). Typically it took
approximately 20 days for the animals to test out. The animals were
treated in groups of 8 to 10 individuals. The highest survival rates
(highest T-C values or increased life span in days) occurred in
individuals in which several doses of doxorubicin loaded nanoparticles
were administered. Table 1 demonstrates the efficacy as well as the
safety of the doxorubicin loaded biopolymer nanoparticles in treating a
primary human brain tumor in athymic mice.

[0105] Targeting molecules can be attached to the crosslinked biopolymer
nanoparticle bioconjugate delivery system to facilitate the interaction
of the delivery system with a tumor, metastatic cancer cell, or other
targeting tissue or organ. This capability was demonstrated by the
following procedures and tests.

[0106] Oxidation of Crosslinked Biopolymer Nanoparticles

[0107] Various different types of functionalities may be introduced onto
the crosslinked biopolymer nanoparticles 10 to provide binding sites for
the aptamer as well as the active agent 22. As described above, various
chemical modification techniques can be employed. A particularly useful
chemical modification is oxidation of starch biopolymers 12 to produce
carboxyl functionalities. To illustrate this, TEMPO-mediated oxidation
was carried out for both crosslinked biopolymer nanoparticles 10
(EcoSphere® 2202 from EcoSynthetix Inc.) as well as for regular
native (unmodified) corn starch. In this method, the starch biopolymer 12
was oxidized with sodium hypochlorite (NaClO) and
2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) radicals, at temperatures
between 0 and 4° C. and pH of 10.8. The degree of oxidation was
controlled by amount of NaClO added. As noted, two types of starch were
used. The first was EcoSphere® starch based nanoparticles and the
second one was regular corn starch purchased from Sigma-Aldrich. The
procedures were as described below.

[0108] In a glass jar, 4 g of EcoSphere® and 80 mL MilliQ water were
added and mixed thoroughly to create a ˜5% dispersion. In a second
jar 4 g of Corn Starch and 80 mL of MilliQ water were added to create a
˜5% solution. The second jar was heated up to above 80° C.
(max 95° C.) under agitation and allowed to fully dissolve.
Subsequently it was cooled to room temperature. Separately, in two 45 mL
tubes 40 mL of water, 38 mg TEMPO, and 508 mg NaBr were added into each
tube (0.01 mol TEMPO per anhydroglucose unit of starch; 0.2 mol NaBr per
anhydroglucose unit of starch), stirred until fully dissolved, and cooled
for 30 minutes in an ice batch. Next the content of one tube was mixed
into each jar. A pH measurement was taken, which initially was 3.8 for
the EcoSphere® jar and 7.4 for the Corn Starch jar. Next 450 μL of
0.5 M NaOH was added to the EcoSphere® jar to reach pH 10.75, and 200
μL of 0.5 M NaOH was added to the Corn Starch jar to reach pH 10.75.
Subsequently, 10 mL of NaClO was added when the pH dropped to around 6-7,
and pH measurements were taken every 10-15 min. As the mixtures continued
to stir and the pH dropped, the color became darker (yellow/orange). A
total of 60 mL NaClO was added and the pH was finally adjusted to 8.0
before the oxidized starch was diluted 1:1 with ethanol. Ethanol
precipitated the modified EcoSphere® nanoparticles and modified
starch and they were harvested by centrifugation and washed by water and
ethanol and finally dried by lyophilization (freeze-drying).

[0109] The oxidized EcoSphere® was characterized by zeta-potential
measure and dynamic light scattering. Zeta measurement showed that the
modified particles carried a negative charge with zeta-potential of -25.5
mV, while unmodified particles were essentially neutral. The size of the
particles appeared to be slightly smaller compared to the non-oxidized
ones (i.e. the NTA Mode was 113 versus 141 nm).

[0110] The color of the final product depended on the pH of the solution
after oxidization. If the pH was too high (higher than 10), a yellow
colored product was obtained. It was found that this color can be removed
by lowering the pH.

[0111] DNA Attachment

[0112] Subsequently, amino-modified and fluorescently labeled DNA was
attached to the starch nanoparticles using
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) as a
coupling agent. The reaction mixture contained 5 μM FAM
(6-carboxyfluorescein) and amino dual labeled DNA, 1-5% COOH-modified
starch, 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer, pH 6.0
and 20 mM freshly prepared EDC was added the last. Agarose gel
electrophoresis was carried out for DNA and DNA-conjugated to
TEMPO-oxidized EcoSphere® nanoparticles. It was found that the gel
fluorescence intensity was more evenly distributed and some of the DNA
migrated more slowly, indicating conjugation to the starch nanoparticles.
In some of the alternative DNA attachment protocols the carboxyl groups
on starch were first activated using N-Hydroxylsuccinimide (NHS) at 5 mM
(1/4 of the amount of EDC) for 15 minutes before adding the DNA. Next
this mixture was allowed to react for several hours. Without intending to
be bound by any particular theory of operation, NHS may help to
facilitate the EDC linking reaction by activating carboxyl groups so it
can react with an amine to form an amide, rather than a salt with an
amine.

[0113] Thus the DNA used in this example, which served as a model compound
for ligand attachment, was successfully attached. The DNA sequence was
5'-FAM-ACG CAT CTG TGA AGA GAA CCT GGG-NH2-3'.

[0114] Attachment of an Aptamer

[0115] An aptamer was attached to EcoSphere® 2202 particles using the
procedure described above. Attachment of the aptamer was confirmed by
laboratory observations of nanoparticle fluorescence. The aptamer was,
AS1411, which is believed to have (as modified) the sequence:
5-Cy3-TTGGTGGTGGTGGTTGTGGTGGTGGTGG-NH2-3' (i.e. AS1411 aptamer with
Cy3 fluorescent tag and amine group). The fluorescent tag, used for
imaging purposes in the diagnostic gel electrophoresis test, can of
course be omitted if needed. However, an additional purpose for the
fluorescent tagging is to facilitate monitoring of the binding and uptake
of the modified nanoparticles by a cell. As for the DNA described above,
the aptamers had an amine modification on the 3' end of the DNA so that
it could be linked using EDC chemistry to carboxyl functionalities on the
nanoparticle.

[0116] Four 200 microliter wells were prepared with cells of a cervical
cancer cell line (HeLa) and given time to culture and grow. Well 1 was
left with only the HeLa cells. Well 2 had unconjugated AS1411 added to
it. Well 3 had EcoSphere® 2202 nanoparticles with conjugated AS1411
added to it. Well 4 had nanoparticles conjugated with a control sequence
added to it. The control sequence has no known affinity for HeLa cells.
The wells were then allowed to culture for a further 48 hours.

[0117] After the 48 hours had elapsed, cells from the wells were washed to
remove any fluorescent marks on any unbound particles external top the
cells. The cells were then observed under a fluorescence microscope.
Fluorescent marks were observed within the cells of well 3 confirming
that the nanoparticle/aptamer conjugate had been taken into the cells.

[0118] Drug Adsorption and Release Studies

[0119] In a dilute aqueous dispersion (e.g. 1-5%) the EcoSphere®
nanoparticles are highly swollen and their density is close to that of
water. As a result, centrifugation and even ultracentrifugation were
ineffective methods to separate the particles from the aqueous dispersion
media. Instead, drug loading was evaluated by way of fluorescence change.
It was found that the adsorption of the anticancer drug doxorubicin (Dox)
was very much improved after modification of the EcoSphere®
nanoparticles with carboxylate groups. Upon adsorption, the fluorescence
of doxorubicin was also quenched by the carboxylated EcoSphere®. This
was clearly visible under the 245 nm excitation in a dark room using a
handheld UV lamp. The fluorescence quenching provides an analytical
method to monitor doxorubicin adsorption.

[0120] To ensure that the observed quenching was not due to a pH effect,
the fluorescence was subsequently compared for the following: doxorubicin
was dissolved at a final concentration of 0.01 mg/mL in unmodified
EcoSphere®, COOH-modified EcoSphere® and buffer (no
EcoSphere®). For each condition, two pH conditions were tested to
contain either 20 mM sodium acetate buffer (pH 5.0) or 20 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, pH
7.6. The final pH was confirmed to be at the intended values.

[0121] Free doxorubicin fluorescence was strong in both pH 5 and 7.6 in
water. Mixing with 1% unmodified EcoSphere® nanoparticle dispersion
induced about 50% fluorescence quenching but mixing with a COOH-modified
EcoSphere® nanoparticle dispersion completely quenched the
fluorescence. This confirmed that COOH-modified EcoSphere® is better
at adsorbing doxorubicin. Without intending to be bound by any particular
theory of operation, this is likely due to electrostatic interactions
with the positively charged doxorubicin. Therefore, tuning the
EcoSphere® charge will allow selective adsorption of various drugs
and in addition provide a way of controlling the release profile.

[0122] Electrokinetic Measurements

[0123] To evaluate the presence of electrostatic charges on the surface of
the particles, the zeta potential of the biopolymer nanoparticles and
TEMPO oxidized biopolymer nanoparticles was determined from the analysis
of the electrokinetic measurements using a Brookhaven ZetaPlus
instrument. The crosslinked starch particles were suspended in a solution
of NaCl ranging from 0.001 M to 0.1 M concentration, and their
electrophoretic mobilities were determined. The electrophoretic
mobilities were converted to zeta (ζ) potentials using the
Smoluchowski expression, which assumes small particles and dilute ion
concentration. The zeta potential of the un-modified starch based
nanoparticles was determined to be close to zero, whereas the zeta
potential of the TEMPO modified biopolymer nanoparticles was determined
to be -25 mV which indicates negatively charged nanoparticles.

[0124] Particle Size Analysis

[0125] The particle size of dispersed starch based nanoparticles and the
TEMPO modified nanoparticles was determined by Nanoparticle Tracking
Analysis (NTA) using an LM 20 tracking analysis device (NanoSight Ltd.)
equipped with a blue laser (405 nm). The device uses a 50 mW laser
operating in the CW mode to illuminate the particles. The light scattered
by the particles is captured using a digital camera and the motion of
each particle is tracked from frame to frame using NanoSight software. A
high speed video is obtained (30 frames per second, average video about
30 s). The trajectories of individual particles are generated from the
video sequence and the mean squared displacement determined for each
particle. Typically at least 20 trajectories are acquired and 250 to 500
sets of trajectories (each set corresponding to an individual particle)
are accumulated in a video sequence. The analysis of the mean squared
displacement is used to calculate the diffusion coefficient and the
hydrodynamic radius (rh) is determined using the Stokes-Einstein
equation. Thus, the diameter of each particle in the sample can be
determined and a true particle size distribution derived. Because a
diffusion coefficient is obtained for each particle in the field of view,
a particle size distribution can be obtained which does not assume a
particular mathematical model as in dynamic laser light scattering (DLS)
analysis.

[0126] Dispersions of biopolymer nanoparticles were prepared using the
following procedure: 1) dry agglomerate EcoSphere® powder was mixed
in water containing 0.4 wt % sodium carbonate ("lite soda ash") based on
dry weight in a Silverson high shear mixer for 15 minutes; the final
concentration of the dispersed biolatex ranged from 0.015 to 0.030%
(w/w); 2) this dispersion was heated to 45° C. for 15 minutes in a
water bath prior to measurement to ensure the agglomerate particles were
fully dispersed into nanoparticles.

Example 5

[0127] Three batches of crosslinked biopolymer nanoparticles 10 with
different degrees of crosslinking were made. A first batch 100 had the
lowest amount of crosslinking, a second batch 102 had a moderate amount
of crosslinking and a third batch 104 had the highest degree of
crosslinking. One manner to assess the degree of crosslinking is to
determine the maximum volume swell ratios, also referred to as the
effective volume factor, as described in Do lk Lee, Steven Bloembergen,
and John van Leeuwen, "Development of New Biobased Emulsion Binders",
PaperCon2010, "Talent, Technology and Transformation", Atlanta, Ga., May
2-5, 2010, the disclosure of which is incorporated herein by reference.
Briefly, crosslinked biopolymer nanoparticles swell under conditions of
extreme dilution with water to achieve the maximum swelling value that is
balanced between their elastic constraint due to their crosslinked
network and the osmotic pressure (see Bloembergen, S., McLennan, I., Lee,
D. I., and van Leeuwen, J., "Paper Binder Performance with Nanoparticle
Biolatex®: EcoSynthetix develops EcoSphere® biolatex for
replacement of petroleum based latex binders", ACFS, Montreal, Jun.
11-13, 2008).

[0128] By measuring the relative viscosity, ηr, at low
concentrations (i.e. low volume fraction) for a latex (a polymer
colloid), one can gather relevant information about the viscosity and
swelling behavior of that colloid. The relative viscosity
(ηr=η/ηo) of a biobased latex binder is obtained by
simply measuring the flow times between two demarcations of a glass
Ubbelohde viscometer for the biobased latex dispersion (η) and for
its dispersion medium (ηo), which is water. Using the Einstein
equation, ηr=1+2.5 f φ, where f is the effective volume
factor and φ is the volume fraction, one can obtain the effective
volume factor (f) that is equal to the maximum volume swelling of
biobased latex nanoparticles at very low concentrations. The first batch
100 had a maximum volume swell ratio of 16.0, the second batch 102 had a
maximum volume swell ratio of 9.33 and the third batch 104 had a maximum
volume swell ratio of 6.67.

[0129] The three batches 100, 102, 104 were loaded with 5% doxorubicin by
mass, as described above, and dispersed in 5 mL of Milli-Q water. The
released doxorubicin was separated from the crosslinked biopolymer
nanoparticles 10 using dialysis with a molecular weight cut off of 25
kDa. Samples were drawn off at various times over 72 hours and the
fluorescence was measured using a fluorescence plate reader.

[0130] As shown in FIG. 12, the initial background was close to zero,
suggesting that little free doxorubicin was present and the loading
capacity of the three batches 100, 102, 104 was high. The first batch 100
demonstrated the fastest rate of doxorubicin release. The third batch 104
released approximately 20% of the drug after 3 days while the first batch
100 released more than 50% of the doxorubicin over the same period.
Without being bound by theory, these results indicate that the level of
crosslinking may be used to control the release profile of the active
agent 22 from the core 14 of the bioconjugate device 30.

Example 6

[0131] The degree, or effect, of multivalent binding between the targeting
molecule 18 and the target cell surface receptors may modulate the
transport of the bioconjugate device 30 into the phospholipid membrane of
the target cell. Samples were prepared with different ratios of
functionalized, crosslinked biopolymer nanoparticles 10 relative to the
targeting molecule 18, in this example the AS1411 aptamer was attached
similar to the approach described above. The concentration of
functionalized crosslinked biopolymer nanoparticles 10 is represented by
(glycosidic) repeating units of glucose. The inventors prepared samples
with the following molar ratios of glucose repeating units to aptamer:
100:1; 500:1; 1000:1; and 5000:1. These samples, free (unattached)
aptamer and a control DNA aptamer were incubated with HeLa cells for 2
hrs. The HeLa cells were seeded and cultured in a CO2 incubator with
5% oxygen in an eight-well slide for two days to achieve a confluence of
-70%. The culture media was DMEM/F-K12 1:1 (Hyclone) with 10% fetal
bovine serum and 1% penicillin and streptomycin. In pairs of wells, we
added to each 20 μL of 10 μM unconjugated AS1411 aptamer, 10 μM
unconjugated control DNA aptamer or 20 μL of 3 mg/mL conjugated SNPs.
These were incubated for a further 2 hours at 37° C. and 5%
CO2. The unbound materials were washed away with phosphate buffered
saline (PBS, from Cellgro).

[0132] The control DNA aptamer was attached in a ratio of 500:1
(glucose:control aptamer) and the control DNA aptamer is not targeted to
recognize or interact with surface features of cancer cells. After
washing the cells to remove unbound and free material, uptake was
quantified using a fluorescence microplate reader. The fluorescence was
normalized to the equivalent dose of free aptamer. As depicted in FIG.
13, the 500:1 sample demonstrated the highest uptake. It is likely that
at ratios at or beyond 1000:1, or possibly beyond about 750:1, the
distance between aptamers was too far to impart a significant multivalent
binding effect. If the aptamers are packed too closely, proper folding
and interaction, for example binding between the bioconjugate device 30
and the cells, may have been hindered.

Example 7

[0133] HeLa cells, cultured as described above, were exposed for 24 hours
to different bioconjugate devices 30 to assess cell viability. Cell
viability was measured with a lactose dehydrogenase (LDH) assay and the
fluorescence was measured at 490 nm using a microplate reader.
Unmodified, crosslinked biopolymer nanoparticles 10 without any loaded
active agent 22 were non-toxic to cells over a range of doses (FIG. 14A).
Bioconjugate devices 30, with the AS1411 attached aptamer, were loaded 5%
by mass with doxorubicin. Different doses of these bioconjugate devices
were given to the cells, and there was increased cell killing with
doxorubicin levels greater than 0.625 μg. Under similar experimental
conditions, 0.625 μg of free doxorubicin (not shown) was less
effective at killing the cells (83% viability, p=9.0 E-4) than 12.5 μg
of loaded bioconjugate device 30 that delivered a total of 0.625 μg of
doxorubicin. Further, the bioconjuguate device 30, with the AS1411
aptamer attached, and no doxorubicin was non-toxic to the cells (not
shown). Without being bound by theory, the inventors attribute these
results to the ability of the bioconjugate device 30 to penetrate the
cell membrane and release the doxorubicin inside of the cells, which is
compared to the passive penetration of the cell membrane by free
doxorubicin.

[0134] The above description and attached figures are intended to
illustrate at least one embodiment of each claim and not to limit any
invention. The invention is defined by the following claims.